Mori, R; (2013) The Tubulin Folding Pathway: Roles of cofactor C/Tbc1 and small GTPase Arl2/Alp41. Doctoral thesis , UCL (University College London).

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Abstract

Supplying a proper amount of correctly folded α/β-tubulin heterodimers is critical for microtubule dynamics. Formation of assembly-competent heterodimers is remarkably complicated at the molecular level, in which the α- and β-tubulins are separately processed in a chaperone-dependent manner. It is known that this sequential step is performed by the tubulin folding cofactor pathway, comprising of a specific set of regulatory proteins - cofactors A to E. Following detailed biochemical studies in vertebrates, studies have been carried out in in vivo models using S. pombe, where the pathway is absolutely essential for viability and each component is evolutionarily conserved. The significance of the data and the possibility of the cofactors playing additional roles led us to study Tbc1, the cofactor C orthologue, which we isolated as a high-copy suppressor of α2-tubulin mutants (atb2-983). Previous work from others and our own lab indicated that Arl2/Alp41 is a GTP binding protein that would be regulated by the GAP, cofactor C/Tbc1. In order to pursue the in vivo roles of Tbc1 and Alp41, we created temperature-sensitive mutants to analyse defective phenotypes, particularly focussing on microtubule structures and dynamics. Interestingly, we have found a range of phenotypes that may be explained by the resulting GTP/GDP state of the G protein. Consistent with Tbc1 being a GAP for Alp41, genetic data supports that Tbc1 negatively regulates Alp41. Biochemical studies have shown that the interactions of these proteins is conserved in fission yeast, and combined with the genetic data we investigated their physiological significance. In addition, we have identified that Arl2/Alp41 also interacts with cofactor D/Alp1 in a GTP/GDP state dependent manner. This led us to an interesting finding that cofactor D/Alp1 possibly has two opposing roles in the cofactor pathway – to assemble and to disassemble microtubules.

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